Crude membrane from High Five insect cells expressing ABCB1 (50 μg) was incubated with 0–5 μM ERK5-IN-1 for 5 min at room temperature in 50 mM Tris-HCl (pH 7.5). [125I]-IAAP (2200 Ci/nmole, 3 nM) was then added and incubated for another 5 min under subdued light. The samples were cross-linked by UV illumination (365 nm) on ice and labeled ABCB1 was immunoprecipitated using the C219 antibody. Finally, samples were subjected to SDS-PAGE using a 7% Tris-acetate NuPAGE gel, dried and exposed to Bio-Max MR film (Eastman Kodak Co., Rochester, NY) at -80 °C for 4 h. The radioactivity incorporated into the transporter protein was quantified using the Storm 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).
Storm 860 phosphorimager system
Manufactured by GE Healthcare
Sourced in United States
The Storm 860 PhosphorImager system is a laboratory equipment used for the detection and analysis of radioactive signals in biological samples. It utilizes a phosphor storage screen to capture the radioactive signal, which is then scanned and digitized for further analysis. The system provides high-sensitivity detection and quantification of radioactive isotopes, making it a useful tool for various life science applications.
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10 protocols using storm 860 phosphorimager system
1
Photoaffinity Labeling of ABCB1
2
Photoaffinity labeling of ABC transporters
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Crude membrane from High Five insect cells expressing ABCB1 or ABCG2 (50 μg protein) was incubated with 0–10 μM alectinib for 5 min at room temperature in 50 mM Tris-HCl (pH 7.5). [125I]-IAAP (2200 Ci nmol−1, 3 nM ) was added, and incubation was continued for another 5 min under subdued light. The samples were then cross-linked by UV illumination (365 nm) on ice. The labeled ABCB1 and ABCG2 were immunoprecipitated using the corresponding antibody. The samples were then subjected to SDS-polyacrylamide gel electrophoresis using a 7% Tris-acetate NuPAGE gel, dried and exposed to Bio-Max MR film (Eastman Kodak Co., Rochester, NY, USA) at −80 °C overnight. The radioactivity incorporated into the transporter protein was quantified using a Storm 860 Phosphor Imager system (Molecular Dynamics, Sunnyvale, CA, USA).39 (link)
3
ABCG2 Protein Cross-Linking Assay
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The crude membrane (50 μg of protein) was separated from MCF7/FLV1000 cells, and 0–10 μM CM082 was added in 50 mM Tris-HCl (pH 7.5) for a 5-min incubation at room temperature. With protection from light, [125I]-IAAP (3 nM; 2,200 Ci/nmol) was added for another 5-min incubation. Radioactive IAAP could cross-link with the ABCG2 protein by ultraviolet illumination (at a wavelength of 365 nm). Specific ABCG2 monoclonal antibody (BXP21) was introduced for immunoprecipitation of the labeled ABCG2. The samples were added to 7% Tris-acetate NuPAGE gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis. After that, they were dried, followed by exposure to Bio-Max MR film (Eastman Kodak, USA) for one night at −8°C. The Storm 860 phosphorimager system (Molecular Dynamics, Sunnyvale, CA, USA) was introduced for radioactivity determination.
4
Photoaffinity Labeling of ABCG2
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Crude membrane from MCF7/FLV1000 cells overexpressing ABCG2 (50 μg protein) was incubated with 0 – 10 μM dacomitinib for 5 min at room temperature in 50 mM Tris-HCl (pH 7.5). Under subdued light, [125I]-IAAP (2200 Ci/nmole, 3 nM) was added and incubation was continued for another 5 min. The samples were then cross-linked by UV illumination (365 nm) on ice. BXP21 antibody was used to immunoprecipitate the labeled ABCG2. The samples were then subjected to SDS-PAGE using a 7% Tris-acetate NuPAGE gel, dried and exposed to Bio-Max MR film (Eastman Kodak Co., Rochester, NY) at -80oC overnight. Radioactivity incorporated into the transporter protein was quantified by using the Storm 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).
5
ABCB1 and ABCG2 Transporter Binding Assay
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Crude membrane from High Five insect cells expressing ABCB1 or ABCG2 (50 μg protein) was incubated with 0 – 5 μM ceritinib for 5 min at room temperature in 50 mM Tris-HCl (pH 7.5). [125I]-IAAP (2200 Ci/nmole, 3 nM) was added and incubation was continued for a further 5 min under subdued light. The samples were then cross-linked by UV illumination (365 nm) on ice. The labeled ABCB1 and ABCG2 was immunoprecipitated using the C219 and BXP21 antibody, respectively. The samples were then subjected to SDS-PAGE using a 7% Tris-acetate NuPAGE gel, dried and exposed to Bio-Max MR film (Eastman Kodak Co., Rochester, NY) at −80°C for 4 h. The radioactivity incorporated into the transporter protein was quantified using the Storm 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).
6
Photoaffinity Labeling of ABCG2 Transporter
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The photoaffinity labeling of ABCG2 with [125I]-IAAP was examined as our previously described33 (link). In brief, crude membrane from ABCG2-overexpressing MCF7/FLV1000 cells (50 μg protein) was incubated with 0–10 μmol/L rociletinib for 5 min at room temperature in 50 mmol/L Tris-HCl (pH 7.5). [125I]-IAAP (2200 Ci/nmole, 3 nmol/L) was added and the mixture was incubated for an additional 5 min under subdued light. The samples were then cross-linked by UV illumination (365 nm) on ice. BXP21 antibody (Abcam, Cambridge, MA, USA) was used to immunoprecipitate the radio-labeled ABCG2. The samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 7% Tris-acetate NuPAGE gel, dried and exposed to Bio-Max MR film (Eastman Kodak Co., Rochester, NY, USA) at −80 °C for 4 h. Radioactivity incorporated into the transporter protein was visualized using the Storm 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA, USA).
7
ABCG2 Protein Crosslinking Assay
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Crude membranes (1 mg protein/mL) from ABCG2-expressing MCF-7 FLV1000 cells were incubated with 0 to 50 μmol/L of Icotinib for 10 min at 21°C to 23°C in 50 mmol/L Tris-HCl (pH 7.5). The photo-crosslinking of ABCG2 with 3 to 6 nmol/L [125I]-IAAP (2,200 Ci/mmol) followed by immunoprecipitation with BXP-21 was done as previously described [40 ]. Immunoprecipitated ABCG2 protein crosslinked with [125I]-IAAP was resolved on a 7% Tris-acetate gel. The incorporation of [125I]-IAAP into the ABCG2 band was quantified using the STORM 860 PhosphorImager system (Molecular Dynamics) and the software ImageQuaNT, as described [40 ].
8
RNA Methylation Assay with CmoB
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The assay was initiated by mixing 10-mM Tris (pH 8.0), 4-mM MgCl2, 140-μM [14CH3]-SAM (Perkin Elmer), 100 μg of total tRNA prepared from cmoB-deficient cells and 2-μM CmoB in 50 μl. In competition assays to measure the effect of Cx-SAM on methylation, 10-μM Cx-SAM was added to the above mixture. An aliquot of 15 μl was removed at 15, 30 and 45 min after initiation of the assay and quenched with 0.5-ml 0.5-M sodium acetate (pH 4.0). RNA was washed with 0.2-M ammonium acetate (pH 6.0) using an MWCO 10-kDa filter by centrifugation. This washing step was repeated two more times to remove unreacted 14C-SAM. RNA was then spotted on a DE81 filter (GE) and air-dried for 20 min. Images of the radioactive tRNA were recorded on a phosphorimaging plate (Molecular Dynamics) for 2 days and analyzed using a Molecular Dynamics Storm 860 PhosphorImager System with ImageQuant software.
9
Photoaffinity Labeling of ABCB1
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Crude membrane from High Five insect cells expressing ABCB1 (50 μg protein) was incubated with 0–5 μmol/L olmtinib for 5 min at room temperature in 50 mmol/L Tris–HCl (pH 7.5). [125I]-IAAP (2200 Ci/nmole, 3 nmol/L) was added and incubation was continued for a further 5 min under subdued light. The samples were then cross-linked by UV illumination (365 nm) on ice. The labeled ABCG2 was immunoprecipitated using the BXP21 antibody. The samples were then subjected to SDS-PAGE using a 7% Tris-acetate NuPAGE gel, dried and exposed to Bio-Max MR film (Eastman Kodak Co., Rochester, NY, USA) at –80 °C for 4 h. The radioactivity incorporated into the transporter protein was quantified using the Storm 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA, USA).
10
ABCG2 Transporter Binding Assay
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The crude membrane from High Five TM insect cells expressing ABCG2 (50 μg protein) was incubated with 0-10 μM of PCI29732 in 50 mM Tris-HCl (pH 7.5) at room temperature for 5 min. After [ 125 I]-IAAP (2200 Ci/nmole, 3 nM) was added, the mixture was incubated for an additional 5 min under subdued light. The samples were then cross-linked by UV illumination (365 nm) on ice. The labeled ABCG2 was immunoprecipitated using the BXP21 antibody (ab3380, Abcam, Cambridge, UK). The samples were then subjected to separation on SDS-polyacrylamide gel electrophoresis (PAGE) using a 7% Tris-acetate NuPAGE gel, followed by drying the gel, and exposure of the gel to Bio-Max MR film (Eastman Kodak Co., Rochester, NY) at -80 o C for 4 hours. The radioactivity incorporated into the ABCG2 band was quantified using the Storm 860 PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).
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